We have developed protocols applying the cellular thermal shift assay coupled with mass spectrometry (MS-CETSA) to investigate cellular dynamics and identify viral host factors during the early stages of alphavirus infection. CETSA exploits the observation that proteins in intact cells undergo temperature-dependent unfolding and precipitation, similar to purified proteins. Following controlled heating, the remaining soluble fraction reflects the amount of folded protein, enabling generation of protein melting curves across the proteome. MS-CETSA enabled detection of proteome-wide changes as early as 2 and 6 hours after infection with Semliki Forest virus in two distinct cell lines, yielding highly concordant results. These analyses revealed several novel aspects of alphavirus replication. We show that activation of the Akt pathway regulates the balance between genomic and subgenomic mRNA translation and modulates nonsense-mediated decay during infection. In addition, we demonstrate that alphavirus nsP3 preferentially interacts with G3BP2 over its paralogue G3BP1. Functional analyses further revealed that G3BP2 alone is sufficient to provide the full proviral activity required for efficient replication of SFV and chikungunya virus, whereas G3BP1 is not. Finally, our data provide evidence that the RNA polymerase II subunit Rpb8 (POLR2H) is targeted by nsP2 to destabilize the RNA polymerase II complex and suppress host transcription. Together, this work establishes MS-CETSA as a powerful platform for identifying early virus-induced changes in cellular protein behaviour at whole-proteome scale.